Drug-resistant P. falciparum malaria is a major public health problem. Molecular markers for most types of drug resistance have been identified and could serve as surveillance tools. However, individuals in high-transmission areas, like sub-Saharan Africa, usually carry many genetically diverse P. falciparum subpopulations ("variants"). Standard PCR methods are incapable of detecting drug-resistance mutations in variants representing <20% of the parasites in an individual host ("minority variants"). Thus, standard assays underestimate the prevalence of patients infected with resistant parasites because they miss patients with resistant minority variants. We have developed Heteroduplex Tracking Assays (HTAs) which can detect drug-resistant minority variants and accurately measure the relative abundance of variant populations. In a Malawi pilot study, we demonstrated a high prevalence of minority variant pfcrt76 mutations (a marker for chloroquine resistance) which were missed by standard PCR. We have now developed HTA's for minority variants at 3 other important loci (dhfr59, dhfr164, dhps540, for antifolate resistance). We will use these assays to (1) determine whether the prevalence of drug-resistant P. falciparum is higher when measured by HTA than by standard PCR;and (2) measure in-host fitness gains and losses of resistant minority variants in drug- treated and untreated individuals. Prevalence of pfcrt76, dhfr59, dhfr164 and/or dhps540 will be measured by HTA and standard PCR cross-sectionally in samples from Malawi, Zambia, Madagascar, and the DR Congo. Pfcrt76 and dhfr164 fitness will be measured in paired enrollment and recrudescent samples from sulfadoxine-pyrimethamine (SP)- treated patients in Malawi and chloroquine-treated patients in Madagascar. The results of this study have important public health implications. The detection of drug-resistant minority variants - with proven fitness - could enable earlier detection of emerging drug- resistance and greater lead-time to plan changes in drug policy. PUBLIC HEALTH RELEVANCE Better surveillance methods for drug-resistant malaria could be of great benefit to malaria control programs. We are trying to develop a method to measure drug-resistant parasites even when they are the minority of parasites in a single host. This could permit the earlier detection of emerging drug resistance.